DESCRIPTION: Recent advances in stem cell biology provide a unique opportunity for researchers to investigate the molecular mechanisms underlying psychiatric and neurological diseases in living neurons derived from the cells of the affected patient. Several laboratories around the world are generating induced pluripotent stem (iPS) cell lines from hundreds of individuals with neurological disease. However, neuronal and glial differentiation protocols may yield heterogeneous cultures, and there may be variability between lines. Furthermore, for many neurological diseases, it is not clear which neuronal or glial subtype(s) to interrogate. We have established and optimized methodologies for directing hiPSCs to a variety of neuronal and glial fates, and we have developed a high throughput methodology to study secretion of analytes from iPSC-derived neuronal and glial cells at the single cell level in a process called microengraving. In this technique, differentiated neurons and glia are plated in nanowells at a density that favors a single cell per well. Wells are sealed from their neighbors with a glass slid coated with antibodies to the analytes of interest. After analyte capture, the slides are incubated with a detection antibody conjugated to a fluorescent tag to detect each, similar to a traditional sandwich ELISA. Slides are scanned and analyzed using standard microarray instrumentation. After removal of the slides, cells remain in their original nanowells and are either fixed and immunostained or else retrieved for gene expression profiling. Here, we aim to advance the development of this technology through the expansion of the platform to allow for the examination of cell-fate specific responses to small molecule treatments (aim 1) and to genetic perturbations (aim 2). If successful, the development of the methodology outlined herein would increase the overall power of the study of iPSC-derived human neurons and glia by allowing for the detection of meaningful results in a subtype of neurons and glia that could otherwise be missed by solely studying a heterogeneous population. One caveat to this methodology is that cells that are isolated from one another may not behave as they would in vivo. In aim 3, we propose to validate the existence and physiological relevance of the subpopulations identified in aims 1 and 2 through targeted proof-of-principle genetic and small molecule interventions performed in vivo in the adult rodent brain. If successful, the developed technology and associated analysis platforms can be readily applied to the study of the secretion of other analytes of interest as well as to other primary and stem cell-derived cell fates.